The present invention relates to methods for identifying and using compounds that inhibit cancer cell metastasis or promote neurite growth and regeneration.
Amoeboid locomotion requires a quasi-metastable state of the adhesion sites, i.e., of the interactions between (i) the cell""s contractile apparatus, (ii) adhesion molecules and other plasma membrane components, and (iii) the growth substratum. FIG. 1 plots in principle the relationship between the strength of cell adhesion to the substratum and cell motility. At the extreme ends of the curve, locomotion is reduced to zero. At these ends, the cell is either too tightly adherent, or it is so loosely attached that force generation against the substratum is impossible. Optimum motility requires an intermediate, dynamic state that facilitates the making and breaking of adhesions as the cell moves. In other words, a factor that increases motility would not be expected to increase or decrease attachment but to facilitate assembly and disassembly of adhesion sites. Knowledge of the attachment mechanism has increased significantly in recent years, but knowledge about detachment has been rudimentary until recently. Both processes must be elucidated to understand the motility of metastatic cancer cells, and the influence of motility factors.
For example, chemorepellents provide important guidance cues for growth cones during nervous system development. These repellents cause developing neurites to change their course of outgrowth away from the repellent source and, thus, play a critical role in pathfinding for growing or regenerating nerves. However, the mechanisms of action of the repellents are not well understood.
The establishment of metastases is a complex, multi-step phenomenon that begins with dissociation of cancer cells from the primary tumor and invasion of surrounding tissues. Although understanding of the metastatic process is incomplete, at least four elements (in addition to continued cell proliferation) have been identified in recent years: (i) changes in cell adhesion molecules, (ii) secretion/surface expression of proteases, (iii) increased cell motility, and (iv) vascularization of primary and secondary tumors. In addition, factors that promote metastatic progression include genetic instability and defects in cell-cell signaling. For example, loss of the NDP kinase-like protein encoded by nm23, a putative metastasis-suppressor gene, seems to result in altered signaling responsiveness, e.g., in motility assays described in MacDonald et al., J. Biol. Chem. 268:25780-25789 (1993) and Kantor et al., Cancer Res. 53:1971-1973 (1993). Motility has been correlated with metastatic potential as reported in Guirguis et al., Nature, 329:261-263 (1987); Partin et al., Cancer Res, 48:6050-6053 (1988); Partin et al., Proc Natl Acad Sci. USA, 86:1254-1258 (1989); Mohler, supra.; and Stearns and Steams, Cancer Metastasis Rev. 12:39-52 (1993).
A growing number of factors contributing to metastastic progression are being identified. The recently identified KAI 1 gene encodes a putative cell adhesion molecule whose expression reduces prostate carcinoma cell motility and metastasis. However, a universal prognostic marker of prostate carcinoma has not been identified to date. Therefore, a thorough understanding of the mechanisms that trigger invasive cancer cell behavior is particularly important for prostate carcinoma.
Vertebrate amoeboid cell systems, such as polymorphonuclear leukocytes, platelets and the nerve growth cone (the pseudopodal, enlarged leading edge of the growing nerve fiber) have been studied in some detail. Pseudopods of locomoting cells are filled with actin microfilaments, and there is considerable knowledge of the components involved in the regulation of polymerization and of force generation in the actin-based cytoskeleton. At so-called focal adhesion sites, the cytoskeleton interacts with the plasma membrane and, via adhesion molecules, with the extracellular matrix or adhesion molecules on neighboring cells. To make locomotion possible, attachment of adhesion molecules to the growth substratum has to be regulated coordinately with the binding of these adhesion molecules, via linker proteins, to the actin cytoskeleton. Numerous proteins are involved in the intracellular interactions. They include, among others, talin, vinculin, Src family non-receptor tyrosine kinases, focal adhesion kinase, certain types of protein kinase C, and the protein kinase substrate, myristoylated alanine-rich C-kinase substrate (MARCKS) (Burridge et al., Ann Rev Cell Biol. 4:487-525 (1988); Jaken et al., J. Cell Biol., 109:697-704 (1989); Luna and Hitt, Science 258:955-964 (1992); Blackshear, J. Biol. Chem., 268:1501-1504 (1993); Schaller and Parsons, Trends Cell Biol. 3:258-262 (1993)).
There are several classes of adhesion molecules. At least in the case of integrins and cadherins, there is evidence that they function not only as adhesion molecules, but also as receptors that signal ligand binding across the membrane. Conversely, external ligand affinity can be modulated by integrin or cadherin phosphorylation on the inside of the cell. Outside-in signaling triggers focal adhesion assembly by a process known to require tyrosine kinase activity. In summary, adhesion sites are distinctive cell organelles comprised of protein assemblies that regulate cell attachment and play an important role in amoeboid motility.
A variety of amoeboid systems, including macrophages, platelets and nerve growth cones, exhibit high activity of cytosolic phospholipase A2 (cPLA2) and generate high levels of cytosolic arachidonic acid (AA). In platelets, cPLA2 is activated via the thrombin receptor. Also, ras-transfected cancer cells with increased motility exhibit increased cPLA2 activity. This suggests a role for cPLA2 and its product, AA, in the regulation of cell motility. The eicosanoid, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) has long been known to affect leukocyte motility and has been implicated in cancer cell attachment. 12-lipoxygenase (12-LOX), is the enzyme that converts AA into 12-hydroperoxyeicosatetraenoic acid, which is reduced spontaneously to 12(S)-HETE. A correlation between metastatic potential and expression levels of 12-LOX has been reported in Honn et al., Cancer Metastasis Rev. 13:365-396 (1994). These observations also implicate AA and HETEs in the regulation of cell attachment and/or motility.
There is considerable interest in eicosanoids as they relate to the prostate because unsaturated fatty acids inhibit steroid 5xcex1 reductase and lowered AA levels have been correlated with increased malignancy of prostate carcinoma cells. In addition, 12-LOX is elevated in advanced-stage human prostate carcinoma.
Results obtained by other laboratories and discussed above were generated in isolation and never synthesized in the manner described herein. Furthermore, the signaling pathway mediating cellular shape and motility responses to thrombin had not been elucidated. In fact, prior to the present invention, functional assays were performed in vivo or in culture with intact cells and monitored a combination of cellular behaviors such as cell adhesion, detachment and motile behavior.
Accordingly, a need exists for assays that can quickly and selectively identify agents that modulate cell adhesion and detachment. The present invention satisfies this need and provides related advantages as well.
The present invention generally relates to methods of identifying agents that modulate the motility of cells. In one aspect, the methods are accomplished by (a) attaching pseudopods, preferably cancer cells or neurite growth cones, on a substratum, (b) exposing the attached pseudopods to a putative agent, and (c) determining the effect of the putative agent on the pseudopods, wherein a significant change in pseudopod attachment indicates the putative agent is an effective repellent agent. The pseudopods arc preferably attached to the substratum by first diluting the pseudopods 1:1 into 2xc3x97modified Krebs buffer containing 22 mM HEPES buffer, pH 7.2 and a reagent to increase osmolarity, preferably 22 mM sucrose, and then by spinning the solid support containing the substratum for a sufficient speed and time to facilitate attachment, preferably about 2000xc3x97g to about 10,000xc3x97g, for up to 60 minutes, and more preferably about 5000xc3x97g for 15 minutes at room temperature.
Various methods for determining the effect of the putative agent on the pseudopods include, for example, monitoring pseudopod detachment, pseudopod elongation, pseudopod retraction, cell extension, or adhesion sites. In one embodiment, the amount of pseudopods detached from the substratum is measured. In these methods, a significant amount of detached pseudopods indicates the usefulness of the putative agent as a repellent. Particularly useful repellents will detach at least 35%, preferably at least 50%, and most preferably at least 90% of the pseudopods from the substratum.
In another aspect of the invention, the methods are accomplished by exposing whole cells or components of cells, such as the pseudopods identified above, to a putative repellent agent. The ability of the putative agent to activate a parameter of the repellent signaling pathway is then determined. The parameters assayed for activation in these methods include cytosolic phospholipase A2 (cPLA2), 12-lipxygenase (12-LOX) or protein kinase C (PKC). Activated cPLA2 is measured by the amount of arachidonic acid (AA) produced after exposure to the putative agent, whereas activated 12-LOX is measured by the amount of either 12-hydroperoxyeicosatetraenoic acid or preferably 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Protein kinase C activation can be measured by the amount of phosphorylated growth cone proteins, such as phosphorylated MARCKS, MacMARCKS, GAP43, or exogenous snythetic substrate (poly)peptides, such as the phosphorylation site domain peptide (PSD).
The invention further relates to methods of identifying neurite-promoting agents that inhibit the ability of an endogenous repellent from affecting cell motility. The methods are accomplished by attaching cellular pseudopods to a substratum, exposing the attached pseudopods to a putative agent in the presence of a known repellent, and thereafter determining the amount of attached or detached pseudopods. A useful neurite-promoting agent will have been identified if at least 75%, preferably at least 85%, and most preferably at least 95% of the pseudopods remain attached to the substratum. Alternatively, the methods can be accomplished by determining whether the putative agent inhibits the activation of a parameter in the repellent signaling pathway, such as cPLA2, 12-LOX or protein kinase C.
The invention also relates to therapeutic methods of using the agents identified in the above assays. The methods include inhibiting cell motility, for example the metastasis of cancer cells, by administering an effective amount of a repellent agent identified in the assays of the present invention. Methods of promoting neurite growth or regeneration are also provided in which a neurite-promoting agent is administered to a patient in need of increased neurite formation.
The invention also provides methods of identifying a repellent receptor in which a thrombin-responsive cancer cell that does not respond to a specific repellent is transfected with a nucleic acid library from a cell that responds to the repellent. The transfected cancer cells are then exposed to the repellent of interest. If a parameter within the repellent signaling pathway is activated in a transfected cell, the novel repellent receptor is thereafter identified. The identification of novel repellent receptors can lead to the production of repellent agonists or antagonists depending on the desired effect.
A further aspect of the invention relates to methods of using the repellent signaling pathway to develop diagnostic tests for the metastatic potential of cancer cells. The methods involve determining the presence of repellents or repellent receptors or determining whether the cancer cells contain a functional signaling pathway. Test samples from surgically removed tumors, tumor biopsies or cell smears can be used in these methods to determine the metastatic potential of the tumor cells. The deficiency of any of these markers provides evidence the tumor cells may have increased metastatic potential.